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Figure legends

Figure 1 Immunohistochemical analysis of ITLN1 in colorectal cancer (CRC) tissue

specimens. (a) ITLN1 immunostaining in the non-neoplastic colonic mucosa and CRC.

ITLN1 staining was observed in the cytoplasm of goblet cells in non-neoplastic colonic

mucosa, whereas ITLN1 staining was reduced in CRC cells (original magnification × 100).

(b) ITLN1 immunostaining in CRC (original magnification × 100). Some ITLN1 staining

was observed in the cytoplasm of CRC cells. (c) Kaplan-Meier plot of the survival of CRC

patients. (d) Prognostic value of ITLN1 in CRC patients and the survival curve as plotted

using the OncoLnc database.

Figure 2 Effects of the inhibition of ITLN1 on cell growth in colorectal cancer (CRC) cells.

(a) Western blotting of ITLN1 in the cell lysates from three CRC cell lines. (b) Western

blotting of ITLN1 in cell lysates from DLD-1 and WiDr transfected with ITLN1 siRNA or

negative control siRNA. β-Actin was included as a loading control. (c, d) Effect of ITLN1

knockdown on the cell growth of DLD-1 (c) and WiDr (d). Cell growth was assessed by an

MTT assay at 1, 2 and 4 days after seeding on 96-well plates. The mean (bars) and standard

deviation (SD; error bars) of three independent experiments are shown. OD, optical density.

*P < 0.05.

Figure 3 Effect of the downregulation of ITLN1 on the epidermal growth factor receptor

(EGFR) signaling pathway. (a, b) Western blotting of ITLN1, EGFR, phospho-EGFR

(pEGFR), Erk1/2, phospho-Erk1/2 (pErk1/2), Akt and phospho-Akt (pAkt) in cell lysates

from DLD-1 (c) and WiDr (d) transfected with ITLN1 siRNA or negative control siRNA.

Katsuya N et al. 20

β-actin was included as a loading control. We used three filters in each cell line. We

confirmed that total proteins are almost the same amounts in all lanes as monitored by βactin. In this figure, representative western blots for β-actin are shown.

Figure 4 Analysis of the correlation between the expression of ITLN1 and colorectal cancer

(CRC)-related molecules in the invasive front. (a) Expression levels of ITLN1 (b–g) CRCrelated molecules, including β-catenin, matrix metalloproteinase 7 (MMP7), CDX2, claudin18, MUC5AC and CD44 were examined. A serial section showed that β-catenin, MMP7,

CDX2, claudin-18, MUC5AC and CD44 at the invasive front were partially adjacent to the

area in which ITLN1 was reduced.

Figure 5 Analysis of the expression of ITLN1 in pre-cancerous lesions of each colorectal

carcinogenesis pathway. (a–d) Immunostaining of adenoma in colorectal polyps (CRPs). (a)

Conventional adenoma low grade, n=32; (b) conventional adenoma high grade (CAHG),

n=64; (c) traditional serrated adenoma (TSA), n=27 and (d) sessile serrated adenoma/polyp

(SSA/P), n=20. (e, f) ITLN1 immunostaining scores in CRP. The graph indicates the

percentage of sections with different scores (negative, weak, moderate and strong).

Katsuya N et al. 21

Supporting Information

Supporting Figure 1 Quantitative reverse transcriptase PCR analysis of intelectin-1

(ITLN1). (a) ITLN1 mRNA expression level in 13 normal tissues and 5 colorectal cancer

(CRC) cell lines. (b) T/N ratio of ITLN1 mRNA level between CRC tissue (T) and

corresponding nonneoplastic mucosa (N) in 20 CRC cases. Underexpression was defined as a

T/N ratio < 1.0. Underexpression of the ITLN1 gene was observed in 16 (80%) of the 20 GC

cases.

Supporting Table 1 Univariate and multivariate analysis of factors influencing survival in 148

patients with colorectal cancer

Katsuya N et al. 1

Table 1. Relationship between ITLN1 expression and clinicopathologic characteristics in the 148

CRC cases

ITLN1 expression

P-value

Reduced (%)

Preserved

≦ 65 (n = 70)

41 (59%)

29

≧ 66 (n = 78)

46 (59%)

32

Female (n = 89)

55 (62%)

34

Male (n = 59)

32 (54%)

27

T1/T2 (n = 25)

11 (44%)

14

T3/T4 (n = 123)

76 (62%)

47

N0 (n = 81)

46 (57%)

35

N1/2/3 (n =67)

41 (61%)

26

M0 (n = 129)

70 (54%)

59

M1 (n = 19)

17 (89%)

Stage I / II (n = 40)

16 (40%)

24

Stage III/IV (n = 108)

71 (66%)

37

Low(Grade1) (n = 98)

48 (49%)

50

High(Grade2/3) (n = 50)

39 (78%)

11

84 (58%)

60

3 (75%)

Age

NS

Sex

NS

T grade

NS

N grade

NS

M grade

0.0017

Stage

0.005

Budding Grade

0.001

Histlogic classification

Well/moderate (n = 144)

Poor/mucinous (n = 4)

P values were calculated with Fisher’s exact test.

NS, not significant.

NS

Katsuya N et al. 2

Table 2. Relationship between ITLN1 expression and cancer related molecules in 126 patients

with colorectal cancer

ITLN1 expression

Reduced (%)

Reserved

Positive (n = 87)

55 (63%)

32

Negative (n = 39)

15 (38%)

24

Positive (n = 40)

28 (70%)

12

Negative (n = 86)

42 (49%)

44

Positive (n = 69)

39 (57%)

29

Negative (n = 58)

31 (53%)

27

Positive (n = 73)

26 (36%)

47

Negative (n = 53)

44 (83%)

Positive (n = 79)

41 (52%)

38

Negative (n = 47)

29 (61%)

18

Positive (n = 67)

44 (65%)

23

Negative (n = 59)

26 (38%)

33

Positive (n = 15)

12 (80%)

Negative (n = 111)

58 (53%)

53

Positive (n = 79)

50 (63%)

29

Negative (n = 47)

20 (42%)

27

Positive (n = 70)

39 (56%)

31

Negative (n = 56)

31 (55%)

25

Positive (n = 78)

45 (57%)

33

Negative (n = 48)

25 (52%)

23

P-value

β-catenin (nuclear localization)

0.012

MMP7

0.026

P53 expression

NS

CDX2

<0.001

MUC2

NS

MUC5AC

0.02

Claudin-18

0.042

CD44

0.027

MLH1

NS

MSH2

P values were calculated with Fisher’s exact test.

NS, Not significant.

NS

Katsuya N et al. 1

Supporting Table 1. Univariate and multivariate analysis of factors influencing survival in 148 patients with

colorectal cancer

Univariate analysis

HR (95% CI)

Multivariate analysis

P-value

HR (95% CI)

P-value

Age

≦ 65

1 (Reference)

≧ 66

1.49 (0.79-2.92)

NS

Sex

Female

1 (Reference)

Male

0.87 (0.45-1.65)

NS

Classification

T grade

5.18 (0.97-5.03)

＜0.0001

2.47 (0.47-2.12)

NS

N grade

4.89 (2.45-10.59)

＜0.0001

2.05 (1.45-8.39)

NS

M grade

5.52 (2.78-10.51)

＜0.0001

2.81 (1.39-5.67)

0.003

Stage I/II

1 (Reference)

＜0.0001

1 (Reference)

0.0041

Stage III/IV

5.08 (0.10-12.04)

Tumor stage

3.34 （1.44-8.36）

Budding Grade

Low (Grade 1)

1 (Reference)

High (Grade 2/3)

3.31 (1.47-7.31)

0.0006

1 (Reference)

NS

1.56 （077-4.47）

ITLN1 expression

Reduced

1 (Reference)

Preserved

2.93 (1.09-4.81)

P values were calculated with Fisher's exact test.

NS, not significant

0.0126

1 (Reference)

2.57 （1.16-5.34）

0.0376

Figure 1

100

ITLN1 preserved n = 61

ITLN1 preserved n = 225

Overall surviｖal

Overall surviｖal (%)

80

60

40

ITLN1 reduced n = 87

Log rank P = 0.0155

20

Hiroshima cohort

Log rank P = 0.01149

ITLN1 reduced n = 223

TCGA cohort

20

40

60

Survival period (months)

80

Survival period (days)

ITLN1 -

- 31.3kDa

β-actin -

- 41kDa

siRNA-2

siRNA-1

Negative

control

siRNA-1

- 31.3kDa

β-actin -

- 41kDa

WiDr

0.120

control

0.100

siRNA1

siRNA2

0.030

0.020

0.080

O.D. 595

O.D. 595

0.040

WiDr

ITLN1 -

DLD-1

0.060

0.050

DLD-1

Negative

control

WiDr

DLD-1

COLO201

siRNA-2

Figure 2

control

siRNA1

siRNA2

* *

0.060

0.040

0.020

0.010

*P<0.05

0.000

DAY0

DAY1

DAY2

DAY4

*P<0.05

0.000

DAY0

DAY1

DAY2

DAY4

Figure 3

DLD-1

WiDr

ITLN1 siRNA

Negative

control

Negative

control

ITLN1 siRNA

ITLN1 -

- 31.3kDa

ITLN1 -

- 31.3kDa

AKT -

- 60kDa

AKT -

- 60kDa

pAKT -

- 60kDa

pAKT -

- 60kDa

- 44.4kDa

ERK-

ERK-

pERK -

- 44.4kDa

EGFR-

- 170kDa

pEGFR -

- 170kDa

β-actin -

- 41kDa

- 44.4kDa

pERK -

- 44.4kDa

EGFR-

- 170kDa

pEGFR -

- 170kDa

β-actin -

- 41kDa

Figure 4

ITLN1

β-catenin

MMP7

CDX2

Claudin-18

MUC5AC

CD44

Figure 5

CALG

SSA/P

CAHG

TSA

Immunostaining intensity(%)

100%

90%

strong

80%

moderate

70%

weak

60%

negative

50%

40%

30%

20%

10%

*P<0.05

0%

Normal

CALG

CAHG

Immunostaining intensity(%)

NS

100%

90%

strong

80%

moderate

70%

weak

60%

negative

50%

40%

30%

20%

10%

*P<0.05

0%

Normal

TSA

SSA/P

Fold difference (T/N)

2.5

1.5

0.5

LoVo

WiDr

COLO-320

COLO-201

DLD-1

Muscle

Spleen

Proatate

Kidney

Pancreas

Liver

Colon

Small intestine

Stomach

Lung

Heart

Spinal cord

ITLN1 mRNA expression level

Supporting Figure 1

Colon cancer cell lines

9 10 11 12 13 14 15 16 17 18 19 20

...